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Recombinant DNA; rat muscle nicotinic acetylcholine receptor; flanking primer; fragment fusion; amplification; nucleotide sequence
Abstract:
The construction of deletions, insertions and point mutations in DNA sequences is a powerful approach to analysing the function and structure of genes and their products. Here, we present a fast and efficient method using the polymerase chain reaction to introduce mutations into cDNAs coding for the alpha-, gamma- and epsilon-subunit of the rat muscle acetylcholine receptor. Two flanking primers and one mutant oligo, in conjunction with supercoiled plasmid DNA and a fragment of the target DNA are sufficient to introduce the mutation by two PCR amplifications. Our method permits directing the location of mutations anywhere in the target gene with a very low misincorporation rate, as no substitution could be detected within 9600 bp. The utility of this approach is demonstrated by the rapid introduction and analysis of eleven mutations into three different cDNAs. Any kind of mutation can be introduced with an efficiency of at least 50%.
